The presence of endogenous phosphorylase kinase and phosphorylase phosphatase in crude extracts of fat bodies from the cockroaches Nauphoeta cinerea and Periplaneta americana is demonstrated in vitro by activation/inactivation of glycogen phosphorylase under appropriate conditions. Fractionation of fat body extracts of both cockroach species on an anion-exchange medium results in the elution of three peaks with phosphorylase activity. According to their AMP dependency these activity peaks are designated as phosphorylase b (inactive without AMP), phosphorylase ab (active without AMP, but several stimulated with AMP) and phosphorylase a (active without AMP). It is shown chromatographically that incubating crude extracts of fat bodies from both cockroaches, under conditions where the phosphorylase kinase is active, results in all phosphorylase b being converted to the ab- or a-form , whereas under conditions where the phosphorylase phosphatase is active all phophorylase a is converted to the ab- or b-form . Endogenous phosphorylase kinase of N. cinerea crude fat body extract can convert vertebrate phosphorylase b into the a-form , and, conversely, vertebrate muscle p hosphorylase kinase and phosphorylase phosphatase, respectively, are able to convert partially purified N. cinerea phosphorylase aborb and the ab- und a-form , respectively. In resting cockroaches most of the phosphorylase activity resides in the b-form and only a small fraction (10% ) in the a-form , whereas between 26% (N . cinerea) and 35% (P. americana) occurs in the ab-form . Injection of endogenous hypertrehalosaemic peptides into N. cinerea (the decapeptide Bld-HrTH ) or P. americana (the two octapeptides Pea-CAH -I and II) causes interconversion of phosphorylase; after injection, mainly (60% ) phosphorylase a is present, while 25% and 15% exists in the ab- und b-form , respectively. Purification of the three phosphorylase forms from N. cinerea is achieved by anion-exchange chromatography on DEAE-Sephacel followed by affinity chromatography on AMP-Sepharose. The final specific activities are 2.1, 6.9 and 27.2 U /mg protein for the a-, ab- und b-form . The molecular mass of the active molecules on gel filtration is between 173,000 and 177,000, and SDS gel electrophoresis reveals a subunit mass of 87,100, suggesting a homodimeric structure for all three form s. Kinetic studies show hyperbolic saturation curves for the substrates glycogen and Pi respectively, with Kᴍ-values of 0.021, 0.019 and 0.073% for glycogen and 8.3, 6.3 and 17.9 mᴍ for Pi (a-, ab- and b-form ). Phosphorylase a exhibits a more or less hyperbolic response to AMP and needs 70 |iM A M P for m axim al stim ulation. The kinetics for the ab- and b-form s are sigm oidal and maximal activities are displayed at about 3 mᴍ (half-maximum activation as calculated from Hill plots are 55 and 280 μᴍ for the ab- und b-form , respectively). Caffeine is a strong inhibitor of the b-form , but has only a slight inhibiting effect (10 -20 % ) on the ab- and a-form in the presence of AMP.
Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation.
